The vertebrate heart contracts spontaneously, but the force and frequency of contration are increased by norepinephrine (NE) release from sympathetic nerves and acetylcholine (ACh) released from parasympathetic nerves. These transmitters act upon several different effector systems including several different kinds of ion channels in the plasma membrane, the sarcoplasmic reticulum, and proteins in the contractile apparatus. The mechanism of action of NE is relatively well understood: NE increases contractility by stimulating adenylate cyclase which in turn activates the cAMP-dependent protein kinase that phosphorylates the appropriate effector proteins. The molecular mechanisms of action of ACh, on the other hand, are less well understood. The overall goal of this research will continue to be to elucidate the molecular mechanisms which underlie neural (particularly parasympathetic) and hormonal control of the heart. We would like to understand how the binding of ACh to receptors is transmitted to different effector systems, the nature of the "second messenger" systems invovled, and the role of each of the effector systems in regulating contraction. In this next 5-year period, we plan to focus heavily upon regulation of the trans-sarcolemmal calcium current (ICa), because this current plays a central role in determining the force of cardiac contraction. In addition, we will pursue experiments on phosphorylation of C-protein. The research will address 4 specific questions. (1) What is the mechanisms of cGMP action on ICa? We have previously shown that intracellular perfusion with cGMP decreases I Ca under certain conditions, and we have hypothesized that this decrease is mediated by a cGMP-stimulated phosphodiesterase. This hypothesis wil be tested extensively. (2) What are the mechanisms of ACh action on ICa? Does ACh act only by inhibiting adenylate cyclase, or are there other modes of ACh action? (3) Does ACh produce its positive inotropic effects on the heart by stimulating phosphoinositide metabolism and increasing ICa? (4) Are ICa and C-protein and troponin-1 phosphorylation controlled coordinately? It is hoped that these studies will provide new insights into neural control of cardiac function.